2. MATERIALS AND METHODS:

2.1 Chemicals:

All the reagents used were of analytical grade obtained from Merck Specialties Pvt. Ltd, Mumbai, India.

2.2 Plant material collection and authentication:

The plant material was collected from the Malegaon city of Dist. Nasik, Maharashtra, in the month of September- 2009 at the time of collection, plant including leaves and flowers were collected. The plant was authenticated by Mr. Arvind S. Dhabe (Herbarium in charge), Assistant Professor of Department of Botany. Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, (M.S.) India. The leaves and flowers part were separated from other parts, washed, cleaned and dried for further use.

2.3 Extraction of phytoconstituents:

Coarsely powdered materials of leaf and flowers (100 g each) were subjected to reflux consecutively with petroleum ether and methanol. Unsaponifiable matters were separated from saponified petroleum ether extract coded as USM-L and USM-F. Dried leaflets (10gm) were exhaustively extracted with methanol coded as MEL and MEF. The Methanolic extract of leaves and flower were fractionated by extraction with ethyl acetate coded as EA-L and EA-F followed by subsequent filtration and evaporation to yield extract.

2.4 Phytochemical screening:

Preliminary phytochemical screening of leaves and flowers were tested for the presence of phytoconstituents [9, 10].

2.5 Separation and isolation of phytoconstituents by chromatographic methods:

Gradient fractionation of USM-L and USM-F were performed by eluting the column with Chloroform : methanol (10:1) followed by methanol over silica gel(60-80#), resulting in the isolation of compounds designated as Compound A and Compound B from fractions no. 9-22 and 32-36, respectively. The separation of isolates confirmed by spraying with vanillin-sulphuric acid reagent. Methanolic extract of leaf and flower was chromatographed by toluene: ethyl acetate: formic acid (5:4:1), followed by spraying with ferric chloride (Table1) resulting in the isolation of compounds designated as Compound C . Ethyl acetate soluble part (from the methanolic flower and leaves extract) was performed by eluting the column with toluene: ethyl acetate : methanol (4:3:3) followed by methanol over silica gel (60-80#), resulting in the isolation of compounds designated as Compound D and Compound E were evidenced in UV light. The extracts were processed by preparative TLC (dimension: 20 cm × 20 cm, Merck, Germany) to isolate the compounds [11,12] .

2.6 Characterization of isolated compounds:

The isolated compounds were characterized with help of physical characters and spectroscopic analysis (UV spectroscopy, IR spectroscopy, Mass spectrometry and NMR spectroscopy).

2.6.1 Spectroscopic characterization:

Different spectroscopic methods were used to elucidate the structure of isolated compound ST. Among the spectroscopic techniques IR, 1H‐NMR, 13C‐NMR and GC‐MS were carried out. The infra red spectrum was recorded on FTIR Perkin Elmer, 1H‐NMR and 13C‐NMR spectra were recorded using CDCl₃as solvent on Bruker Advance II 400 NMR spectrometer, and GC‐MS spectra were recorded at high resolution on a mass spectrometer (Perkin Elmer Auto system) at SAIF, Indian Institute of Technology, Powai, Mumbai the data are given in m/z values.

3. Results and Discussion:

3.1 Phytochemical screening:

A qualitative phytochemical analysis was performed for the presence of carbohydrate, protein, steroid, flavonoid and tannins Table 1.

3.2 Separation and analysis:

Unsaponifiable matter of petroleum extract of leaves and flower resulted in column chromatographic separation of compound A (β-sitosterol) and compound B (Lupeol)(Table 1). Methanolic extract of leaves and flower resulted compound C (Gallic acid). Ethyl acetate soluble fraction(from methanolic extract) resulted compound D (Quercetin)and compound E(Rutin)(Table 2) and isolated by preparative thin layer chromatography.

3.3 Characterization of phytoconstituents:

3.3.1 Characterization of compound A:

Crystalline white powder, soluble in petroleum ether, acetone and alcohol; m.p. 147ºC, UV (MeOH) max: 211.4 nm. Compound A showed positive Lieberman-Burchard test for steroid. The IR spectrum revealed the presence of hydroxyl group at 3426.3 cm^-1and C=C at 1648, 1636 cm^-1. The ¹H NMR showed signals at δ1.14, 1.26, 0.91, 1.01, 0.97 and 0.93 for methyl protons. The olefinic resonance was observed at δ 5.34 as multiplet, while the carbinylic proton centered at 3.52 as a multiplet. The ¹³C NMR showed the signals at 140.7 and 121.6, which confirmed the presence of an olefinic bond between C-5 and C-6. The molecular ion peak was deduced from MS at m/z 415 [M+H]⁺corresponding to the molecular formula C₂₉H₅₀O. A comparative study of its spectroscopy data with the literature revealed that compound A was identified phytosterol as β- sitosterol.

3.3.2 Characterization of compound B:

Compound B showed positive Lieberman-Burchard test for steroid. Compound B is isolated as colourless crystals and showed m.p. at 214-215 ⁰C. The IR spectrum revealed the presence of hydroxyl group at 3306 cm^-1, C-O at 1732, 1637 cm^-1. The ¹H NMR showed signals at δ(ppm) 4.566, 3.2, 1.9, 1.2, 0.90 for methyl protons. The olefinic resonance was observed at δ 5.34 as multiplet, while the carbinylic proton centered at 7.261 as a multiplet. The molecular ion peak was deduced from MS at m/z 426 [M+H]⁺corresponding to the molecular formula C₃₄H₅₂O (calcd. 439). A comparative study of its spectroscopy data with the literature revealed that compound B was identified phytosterols as Lupeol.

3.3.3 Characterization of compound C

Compound C is isolated as white needle crystals. The UV spectrum of compound C showed two major absorption peaks at 219 nm. The positive FAB-MS gives a molecular ion peak at m/z 178.1 which is compatible with the molecular formula C₇H₆O₅. IR absorption bands at 3277, 1664, 1597 and 1166 were consistent with the presence of hydroxyl, carbonyl, aromatic ring and ether groups respectively. The ¹H NMR spectra showed two doublet at δ 7.97 and δ 6.79 which may be assigned for the protons of B ring. Signals at δ 6.08 and δ 6.29 were due to the protons attached to C-6 and C-8 respectively. The ¹³C NMR spectrum showed the signals in the downfield indicating the aromatic nature of the carbon. The signal at δ 177.30 may be assigned for the carboxylic carbon. Based on the spectral

data and comparison of the data given in the literature, the structure of compound E was identified as Gallic acid.

Gallic acid

3.3.4 Characterization of compound D

Compound D is isolated as a yellow amorphous powder. The UV spectrum of compound C showed two major absorption peaks at 256 and 372 nm characteristic for flavonols. The positive FAB-MS of this compound gave a quasi-molecular ion peak [M+H]⁺ at m/z 303.0, compatible with the molecular formula C₁₅H₁₀O₇ (Calcd.302.23). Its UV absorptions in MeOH were consistent with the presence of a 3, 5, 7, 3’, 4’ pentahydroxyflavone structure. IR absorption band at 3296, 1659, 1596 and 1169 cm^-1 were consistent with the presence of hydroxyl, carbonyl, aromatic ring and ether groups respectively. The ¹H- and ¹³C-NMR spectra of compound G exhibited resonances due to aromatic systems. In the ¹H-NMR spectrum of G, doublet at δ 7.63 may be assigned to H-2’ proton of B ring. The doublet at δ 7.52 and δ 6.77 could be assigned to H-6’ and H-5’ proton of B ring respectively. The signals at δ 6.08 and δ 6.29 were due to the protons attached to C-6 and C-8 respectively. The ¹³C NMR spectrum of G showed the presence of 15 aromatic carbon signals. The signal at δ 177.33 indicated the presence of carbonyl carbon. Based on the NMR data and comparison of the data given in the literature, the structure of compound G was identified as Quercetin.

Quercetin [2-(3, 4-Dihydroxyphenyl)-3, 5, 7-trihydroxy-4H-1benzopyran-4-one]

3.3.5 Characterization of compound E

Compound E is isolated as pale yellow amorphous powder. The UV spectrum of compound showed two major absorption peaks at 2 and 357 nm. These peaks are characteristic for flavonols which show major absorption peaks in the region 200-400 nm. These two peaks are commonly referred to as band I (300-380 nm) and band II (240-280 nm). Band I is considered to be associated with the absorption due to the B-ring (cinnamoyl system) and band II with the absorption involving the A-ring, benzoypyran system. The positive FAB-MS gives a molecular ion peak at m/z 610 which is compatible with the molecular formula C₁₅H₁₀O_{6
ribose sugar .} IR absorption bands at 3277, 1664, 1597 and 1166 were consistent with the presence of hydroxyl, carbonyl, and aromatic ring and ether groups respectively. The ¹H NMR spectra showed two doublet at δ 7.97 and δ 6.79 which may be assigned for the protons of B ring. Signals at δ 6.08 and δ 6.29 were due to the protons attached to C-6 and C-8 respectively. The ¹³C NMR spectrum showed the signals in the downfield indicating the aromatic nature of the carbon. The signal at δ 177.30 may be assigned for the carboxylic carbon. Based on the spectral data and comparison of the data given in the literature, the structure of compound E was identified as

Rutin.

[3-o-rutinose,5,7dihydroxyphenyl-2(3’4’-dihydroxyphenyl)-4H-1benzopyran-4-one]

4. DISSCUSSION

4.1 Structural elucidation of isolated compound A (β- sitosterol):

λ _maxfrom UV spectrum indicated the absence of conjugation and chromophore. FT-IR spectra resulted in presence of functional groups hydroxyl (-OH) stretch, C-H stretch of alkenes and C=C stretch for cycloalkenes. ¹H NMR and ¹³C NMR showed aliphatic protons and hydroxyl proton and presence of 29 carbons in structure. The molecular weight of compound (m/e 415.1)corresponding to the molecular formula C₂₉H₅₀O it was confirmed by mass spectrum. A comparative study of its spectroscopy data with the literature revealed that compound A is steroidal triterpenoid β- sitosterol.

4.2 Structural elucidation of isolated compound B (Lupeol):

λ _maxfrom UV spectrum indicated the absence of conjugation and chromophore. FT-IR spectra resulted in presence of functional groups hydroxyl (-OH) stretch, C-H stretch of alkenes and C=C stretch for cycloalkenes. ¹H NMR and ¹³C NMR showed aliphatic protons and hydroxyl proton and presence of 30 carbons in structure. The molecular weight of compound (m/e 426)corresponding to the molecular formula C₃₀H₅₂O it was confirmed by mass spectrum. A comparative study of its spectroscopy data with the literature revealed that compound B is Pentacyclic triterpenoid Lupeol.

4.3 Structural elucidation of isolated compound C (Gallic acid):

λ _maxfrom UV spectrum indicated the absence of conjugation and chromophore. FT-IR spectra resulted in presence of functional groups hydroxyl (-OH) stretch, C-H stretch of alkenes and aliphatic carbon, C=O stretch and C-O strech,. ¹H NMR and ¹³C NMR showed aromatic protons and hydroxyl proton and presence of seven carbons in structure. The molecular weight of compound (m/e 179)corresponding to the molecular formula C₇H₆O₅ it was confirmed by mass spectrum. A comparative study of its spectroscopy data with the literature revealed that compound E is phenolic compound Gallic acid.

4.4 Structural elucidation of isolated compound D (Quercetin):

λ _maxfrom UV spectrum indicated the presence of conjugation and two chromophore which is specific character of flavonoids. FT-IR spectra resulted in presence of functional groups hydroxyl (-OH) stretch, C-H stretch of alkenes and C=O stretch for lactone and aromatic benzonoid ring. ¹H NMR and ¹³C NMR showed aromatic protons and hydroxyl proton and presence of 15 carbons in structure. The molecular weight of compound (m/e 302.24)corresponding to the molecular formula C₁₅H₁₀O₇ it was confirmed by mass spectrum. A comparative study of its spectroscopy data with the literature revealed that compound C is Phenyl propanoid flavanol Quercetin.

4.5 Structural elucidation of isolated compound E (Rutin):

λ _maxfrom UV spectrum indicated the presence of conjugation and two chromophore which is specific character of flavonoids. FT-IR spectra resulted in presence of functional groups hydroxyl (-OH) stretch, C-H stretch of alkenes and C=O stretch for lactone and aromatic benzonoid ring. ¹H NMR and ¹³C NMR showed aromatic protons and hydroxyl proton and presence of 27carbons in structure. The molecular weight of compound (m/e610.52)corresponding to the molecular formula C₂₇H₃₀O₁₆ it was confirmed by mass spectrum. A comparative study of its spectroscopy data with the literature revealed that compound D is Phenyl propanoid flavanol Rutin.

5. CONCLUSION:

From the above finding β-sitosterol, Lupeol were isolated from petroleum ether extract and gallic acid, Rutin , Quercetin were isolated from methanolic extract of leaves and flowers of Quiaqualis indica and chemical structure elucidated respectively. It was carried out by means of various physical(solvent extraction and chromatography ) and spectroscopical technique. β-sitosterol and lupeol are phytosterols, it is already reported that phytosterol found in a variety of plants and having anti-tumor, anti-inflammatory, immunomodulators and anti-microbial activities. Phenolic compounds(gallic acid) are commonly found in both edible and non-edible plants and they have been reported to have multiple biological effects, including antioxidant property and flavonoids can directly react with superoxide anions and lipid peroxyl radical and consequently inhibit or break the chain of lipid peroxidation. This radical scavenging activity of extracts could be related to the antioxidant nature of polyphenols or flavonoids, thus contributing to their electron/hydrogen donating ability. However, this claim demands further study of pharmacological activity, isolation of active components responsible and clinical studies to establish its safety and efficacy in above mentioned therapeutic properties.

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Received on 06.06.2012

Modified on 02.07.2012

Accepted on 14.07.2012

Research Journal of Pharmacognosy and Phytochemistry. 4(4): July- August 2012, 229-233